PROJECT SUMMARY/ABSTRACT The primary goal of this proposal is to elucidate the mechanism of a newly discovered mutational process called chromothripsis. Chromothripsis generates rapid karyotype evolution in cancer, congenital disease, and other contexts. Chromothripsis is characterized by extensive genomic rearrangements and an oscillating pattern of DNA copy number levels, all surprisingly restricted to one or a few chromosomes. We have made significant recent progress in defining a mechanism for chromothripsis. We showed that intact chromosomes missegregated into aberrant cancer-associated nuclear structures called micronuclei (MN) develop extensive DNA damage. This led us to propose that the physical isolation of chromosomes into MN could cause chromothripsis. Recently, we developed a method (Look-Seq) to combine live cell imaging and single cell genome sequencing, enabling us to recreate chromothripsis in the laboratory and directly demonstrate that it can originate from disrupted MN. We established that chromothripsis could arise by fragmentation and reassembly of MN chromosomes. These fragments can also circularize, potentially providing a mechanism for forming double minute chromosomes, major vehicles for oncogene amplification in cancer. The Look-Seq approach now positions the laboratory to attack the key mechanistic questions in the field: the timing and order of chromosome fragmentation and reassembly, the mechanism of MN chromosome fragmentation; and how it is reassembled. We also propose a series of experiments to define the contribution of DNA replication errors in generating localized chromosome rearrangements. Finally, we propose experiments to elucidate the mechanistic basis for two major sources of oncogene amplification in cancer.